Proteases from gram-positive organisms

ABSTRACT

The present invention relates to the identification of novel serine proteases in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for the  Bacillus subtilis  serine proteases SP1, SP2, SP3, SP4 and SP5. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding SP1, SP2, SP3, SP4 and SP5. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides a cleaning composition comprising a serine protease of the present invention.

This is a Divisional of U.S. patent application Ser. No. 09/462,845,filed on Jan. 13, 2000 now U.S. Pat. No. 6,723,550, which claimspriority benefit to PCT/US98/14647, filed Jul. 14, 1998, and EP97305232.7, filed Jul. 15, 1997.

FIELD OF THE INVENTION

The present invention relates to serine proteases derived fromgram-positive microorganisms. The present invention provides nucleicacid and amino acid sequences of serine protease 1, 2, 3, 4 and 5identified in Bacillus. The present invention also provides methods forthe production of serine protease 1, 2, 3, 4 and 5 in host cells as wellas the production of heterologous proteins in a host cell having amutation or deletion of part or all of at least one of the serineproteases of the present invention.

BACKGROUND OF THE INVENTION

Gram-positive microorganisms, such as members of the group Bacillus,have been used for large-scale industrial fermentation due, in part, totheir ability to secrete their fermentation products into the culturemedia. In gram-positive bacteria, secreted proteins are exported acrossa cell membrane and a cell wall, and then are subsequently released intothe external media usually maintaining their native conformation.

Various gram-positive microorganisms are known to secrete extracellularand/or intracellular protease at some stage in their life cycles. Manyproteases are produced in large quantities for industrial purposes. Anegative aspect of the presence of proteases in gram-positive organismsis their contribution to the overall degradation of secretedheterologous or foreign proteins.

The classification of proteases found in microorganisms is based ontheir catalytic mechanism which results in four groups: the serineproteases; metalloproteases; cysteine proteases; and aspartic proteases.These categories can be distinguished by their sensitivity to variousinhibitors. For example, the serine proteases are inhibited byphenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate(DIFP); the metalloproteases by chelating agents; the cysteine enzymesby iodoacetamide and heavy metals and the aspartic proteases bypepstatin. The serine proteases have alkaline pH optima, themetalloproteases are optimally active around neutrality, and thecysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks, Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press, NewYork).

Proteolytic enzymes that are dependent upon a serine residue forcatalytic activity are called serine proteases. As described in Methodsin Enzymology, vol. 244, Academic Press, Inc. 1994, page 21, serineproteases of the family S9 have the catalytic residue triad “Ser-Asp-Hiswith conservation of amino acids around them.

SUMMARY OF THE INVENTION

The present invention relates to the unexpected discovery of fiveheretofore unknown or unrecognized S9 type serine proteases found inuncharacterized translated genomic nucleic acid sequences of Bacillussubtilis, designated herein as SP1, SP2, SP3, SP4 and SP5 having thenucleic acid and amino acid as shown in the Figures. The presentinvention is based, in part, upon the presence the amino acid triadS-D-H in the five serine proteases, as well as amino acid conservationaround the triad. The present invention is also based in part upon theheretofore uncharacterized or unrecognized overall amino acidrelatedness that SP1, SP2, SP3, SP4 and SP5 have with the serineprotease dipeptidyl-amino peptidase B from yeast (DAP) and with eachother.

The present invention provides isolated polynucleotide and amino acidsequences for SP1, SP2, SP3, SP4 and SP5. Due to the degeneracy of thegenetic code, the present invention encompasses any nucleic acidsequence that encodes the SP1, SP2, SP3, SP4 and SP5 deduced amino acidsequences shown in FIGS. 2A-2B-FIG. 6, respectively.

The present invention encompasses amino acid variations of B. subtilisSP1, SP2, SP3, SP4 and SP5 disclosed herein that have proteolyticactivity. B. subtilis SP1, SP2, SP3, SP4 and SP5, as well asproteolytically active amino acid variations thereof, have applicationin cleaning compositions. In one aspect of the present invention, SP1,SP2, SP3, SP4 and SP5 obtainable from a gram-positive microorganism areproduced on an industrial fermentation scale in a microbial hostexpression system. In another aspect, isolated and purified SP1, SP2,SP3, SP4 or SP5 obtainable from a gram-positive microorganism is used incompositions of matter intended for cleaning purposes, such asdetergents. Accordingly, the present invention provides a cleaningcomposition comprising at least one of SP1, SP2, SP3, SP4 and SP5obtainable from a gram-positive microorganism. The serine protease maybe used alone in the cleaning composition or in combination with otherenzymes and/or mediators or enhancers.

The production of desired heterologous proteins or polypeptides ingram-positive microorganisms may be hindered by the presence of one ormore proteases which degrade the produced heterologous protein orpolypeptide. Therefore, the present invention also encompassesgram-positive microorganism having a mutation or deletion of part or allof the gene encoding SP1, SP2, SP3, SP4 and/or SP5, which results in theinactivation of their proteolytic activity, either alone or incombination with deletions or mutations in other proteases, such as apr,npr, epr, mpr for example, or other proteases known to those of skill inthe art. In one embodiment of the present invention, the gram-positiveorganism is a member of the genus Bacillus. In another embodiment, theBacillus is Bacillus subtilis.

In another aspect, the gram-positive microorganism host having one ormore deletions or mutations in a serine protease of the presentinvention is further genetically engineered to produce a desiredprotein. In one embodiment of the present invention, the desired proteinis heterologous to the gram-positive host cell. In another embodiment,the desired protein is homologous to the host cell. The presentinvention encompasses a gram-positive host cell having a deletion orinterruption of the naturally occurring nucleic acid encoding thehomologous protein, such as a protease, and having nucleic acid encodingthe homologous protein or a variant thereof re-introduced in arecombinant form. In another embodiment, the host cell produces thehomologous protein. Accordingly, the present invention also providesmethods and expression systems for reducing degradation of heterologousor homologous proteins produced in gram-positive microorganismscomprising the steps of obtaining a Bacillus host cell comprisingnucleic acid encoding said heterologous protein wherein said host cellcontains a mutation or deletion in at least one of the genes encodingSP1, SP2, SP3, SP4 and SP5; and growing said Bacillus host cell underconditions suitable for the expression of said heterologous protein. Thegram-positive microorganism may be normally sporulating ornon-sporulating.

The present invention provides methods for detecting gram positivemicroorganism homologs of B. subtilis SP1, SP2, SP3, SP4 and SP5 thatcomprises hybridizing part or all of the nucleic acid encoding B.subtilis SP1, SP2, SP3, SP4 and SP5 with nucleic acid derived fromgram-positive organisms, either of genomic or cDNA origin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows the DNA (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) for SP1 (YUXL).

FIGS. 2A-2B show an amino acid alignment between DAP (dap2_yeast) (SEQID NO:3) and SP1 (YUXL). For FIGS. 2A-2B, 3 and 4, the amino acid triadS-D-H is indicated.

FIG. 3 shows an amino acid alignment between SP1 (YUXL) (SEQ ID NO:2)and SP2 (YTMA) (SEQ ID NO:5).

FIG. 4 shows and amino acid alignment between SP1 (YUXL) (SEQ ID NO:2)and SP3 (YITV) (SEQ ID NO:7).

FIG. 5 shows and amino acid alignment between SP1 (YUXL) (SEQ ID NO:2)and SP4 (YQKD) (SEQ ID NO:9).

FIG. 6 shows and amino acid alignment between SP1 (YUXL) (SEQ ID NO:2)and SP5 (CAH) (SEQ ID NO:10).

FIGS. 7A-7B shows the DNA (SEQ ID NO:4) and deduced amino acid sequencefor SP2 (YTMA) (SEQ ID NO:5).

FIGS. 8A-BB shows the DNA (SEQ ID NO:6) and deduced amino acid sequencefor SP3 (YITV) (SEQ ID NO:7).

FIGS. 9A-9B shows the DNA (SEQ ID NO:8) and deduced amino acid sequencefor SP4 (YQKD) (SEQ ID NO:9).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions—As used herein, the genus Bacillus includes all membersknown to those of skill in the art, including but not limited to B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B.lautus and B. thuringiensis.

The present invention encompasses novel SP1, SP2, SP3, SP4 and SP5 fromgram positive organisms. In a preferred embodiment, the gram-positiveorganisms is a Bacillus. In another preferred embodiment, thegram-positive organism is Bacillus subtilis. As used herein, “B.subtilis SP1 (YuxL) refers to the DNA and deduced amino acid sequenceshown in FIGS. 1A-1C and FIGS. 2A-2B; SP2 (YtmA) refers to the DNA anddeduced amino acid sequence shown in FIGS. 7A-7B and FIG. 3; SP3 (YitV)refers to the DNA and deduced amino acid sequence shown in FIGS. 8A-8Band FIG. 4; SP4 (YqkD) refers to the DNA and deduced amino acid sequenceshown in FIGS. 9A-9B and FIG. 5; and SP5 (CAH) refers to the deducedamino acid sequence shown in FIG. 6. The present invention encompassesamino acid variations of the B. subtilis amino acid sequences of SP1,SP2, SP3, SP4 and SP5 that have proteolytic activity. Such proteolyticamino acid variants can be used in cleaning compositions.

As used herein, “nucleic acid” refers to a nucleotide or polynucleotidesequence, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homolog” as used herein refers to anovel gram-positive microorganism polynucleotide that has at least 80%,at least 90% and at least 95% identity to B. subtilis SP1, SP2, SP3, SP4or SP5, or which is capable of hybridizing to B. subtilis SP1, SP2, SP3,SP4 or SP5 under conditions of high stringency and which encodes anamino acid sequence having serine protease activity.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in a gram-positive host cell.Examples of heterologous proteins include enzymes such as hydrolasesincluding proteases, cellulases, amylases, carbohydrases, and lipases;isomerases such as racemases, epimerases, tautomerases, or mutases;transferases, kinases and phophatases. The heterologous gene may encodetherapeutically significant proteins or peptides, such as growthfactors, cytokines, ligands, receptors and inhibitors, as well asvaccines and antibodies. The gene may encode commercially importantindustrial proteins or peptides, such as proteases, carbohydrases suchas amylases and glucoamylases, cellulases, oxidases and lipases. Thegene of interest may be a naturally occurring gene, a mutated gene or asynthetic gene.

The term “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in a gram-positive host cell. The inventionincludes host cells producing the homologous protein via recombinant DNAtechnology. The present invention encompasses a gram-positive host cellhaving a deletion or interruption of the nucleic acid encoding thenaturally occurring homologous protein, such as a protease, and havingnucleic acid encoding the homologous protein, or a variant thereofre-introduced in a recombinant form. In another embodiment, the hostcell produces the homologous protein.

As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

As used herein, the phrase “proteolytic activity” refers to a proteinthat is able to hydrolyze a peptide bond. Enzymes having proteolyticactivity are described in Enzyme Nomenclature, 1992, edited WebbAcademic Press, Inc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The unexpected discovery of the serine proteases SP1, SP2, SP3, SP4 andSP5 in B. subtilis provides a basis for producing host cells, expressionmethods and systems which can be used to prevent the degradation ofrecombinantly produced heterologous proteins. In a preferred embodiment,the host cell is a gram-positive host cell that has a deletion ormutation in the naturally occurring serine protease said mutationresulting in the complete deletion or inactivation of the production bythe host cell of the proteolytic serine protease gene product. Inanother embodiment of the present invention, the host cell isadditionally genetically engineered to produced a desired protein orpolypeptide.

It may also be desired to genetically engineer host cells of any type toproduce a gram-positive serine protease SP1, SP2, SP3, SP4 or SP5. Suchhost cells are used in large scale fermentation to produce largequantities of the serine protease which may be isolated or purified andused in cleaning products, such as detergents.

I. Serine Protease Nucleic Acid and Amino Acid Sequences

The SP1, SP2, SP3 and SP4 polynucleotides having the sequences as shownin the Figures encode the Bacillus subtilis serine SP1, SP2, SP3, andSP4. As will be understood by the skilled artisan, due to the degeneracyof the genetic code, a variety of polynucleotides can encode theBacillus SP1, SP2, SP3, SP4 and SP5. The present invention encompassesall such polynucleotides.

The present invention encompasses novel SP1, SP2, SP3, SP4 and SP5polynucleotide homologs encoding gram-positive microorganism serineproteases SP1, SP2, SP3, SP4 and SP5, respectively, which have at least80%, or at least 90% or at least 95% identity to B. subtilis as long asthe homolog encodes a protein that has proteolytic activity.

Gram-positive polynucleotide homologs of B. subtilis SP1, SP2, SP3, SP4or SP5 may be obtained by standard procedures known in the art from, forexample, cloned DNA (e.g., a DNA “library”), genomic DNA libraries, bychemical synthesis once identified, by cDNA cloning, or by the cloningof genomic DNA, or fragments thereof, purified from a desired cell.(See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred sourceis from genomic DNA. Nucleic acid sequences derived from genomic DNA maycontain regulatory regions in addition to coding regions. Whatever thesource, the isolated serine protease gene should be molecularly clonedinto a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the SP1, SP2, SP3, SP4 or SP5 may be accomplished ina number of ways. For example, a B. subtilis SP1, SP2, SP3, SP4 or SP5gene of the present invention or its specific RNA, or a fragmentthereof, such as a probe or primer, may be isolated and labeled and thenused in hybridization assays to detect a gram-positive SP1, SP2, SP3,SP4 or SP5 gene. (Benton, W. and Davis, R., 1977, Science 196: 180;Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. USA72:3961). Those DNA fragments sharing substantial sequence similarity tothe probe will hybridize under stringent conditions.

Accordingly, the present invention provides a method for the detectionof gram-positive SP1, SP2, SP3, SP4 or SP5 polynucleotide homologs whichcomprises hybridizing part or all of a nucleic acid sequence of B.subtilis SP1, SP2, SP3, SP4 or SP5 with gram-positive microorganismnucleic acid of either genomic or cDNA origin.

Also included within the scope of the present invention aregram-positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B. subtilis SP1, SP2, SP3, SP4or SP5 under conditions of intermediate to maximal stringency.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.) incorporated herein by reference, andconfer a defined “stringency” as explained below.

“Maximum stringency” typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y.). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from B. subtilis SP1,SP2, SP3, SP4 or SP5 preferably about 12 to 30 nucleotides, and morepreferably about 20-25 nucleotides can be used as a probe or PCR primer.

The B. subtilis amino acid sequences SP1, SP2, SP3, SP4 and SP5 (shownin FIGS. 2A-2B through FIG. 6) were identified via a FASTA search ofBacillus subtilis genomic nucleic acid sequences. B. subtilis SP1 (YuxL)was identified by its structural homology to the serine protease DAPclassified as an S9 type serine protease, designated in FIGS. 2A-2B as“dap2_yeast”. As shown in FIGS. 2A-2B, SP1 has the amino acid dyad“S-D-H” indicated. Conservation of amino acids around each residue isnoted in FIGS. 2A-2B through FIG. 6. SP2 (YtmA); SP3 (YitV); SP4 (YqkD)and SP5 (CAH) were identified upon by their structural and overall aminoacid homology to SP1 (YuxL). SP1 and SP4 were described in Parsot andKebayashi, respectively, but were not characterized as serine proteasesor serine proteases of the S9 family.

II. Expression Systems

The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram-positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram-positive SP1, SP2, SP3, SP4 orSP5 such that the respective activity is deleted. In an alternativeembodiment of the present invention, a gram-positive microorganism isgenetically engineered to produce a serine protease of the presentinvention.

Inactivation of a Gram-positive Serine Protease in a Host Cell

Producing an expression host cell incapable of producing the naturallyoccurring serine protease necessitates the replacement and/orinactivation of the naturally occurring gene from the genome of the hostcell. In a preferred embodiment, the mutation is a non-revertingmutation.

One method for mutating nucleic acid encoding a gram-positive serineprotease is to clone the nucleic acid or part thereof, modify thenucleic acid by site directed mutagenesis and reintroduce the mutatednucleic acid into the cell on a plasmid. By homologous recombination,the mutated gene may be introduced into the chromosome. In the parenthost cell, the result is that the naturally occurring nucleic acid andthe mutated nucleic acid are located in tandem on the chromosome. Aftera second recombination, the modified sequence is left in the chromosomehaving thereby effectively introduced the mutation into the chromosomalgene for progeny of the parent host cell.

Another method for inactivating the serine protease proteolytic activityis through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded serine protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

Deletion of the naturally occurring gram-positive microorganism serineprotease can be carried out as follows. A serine protease gene includingits 5′ and 3′ regions is isolated and inserted into a cloning vector.The coding region of the serine protease gene is deleted form the vectorin vitro, leaving behind a sufficient amount of the 5′ and 3′ flankingsequences to provide for homologous recombination with the naturallyoccurring gene in the parent host cell. The vector is then transformedinto the gram-positive host cell. The vector integrates into thechromosome via homologous recombination in the flanking regions. Thismethod leads to a gram-positive strain in which the protease gene hasbeen deleted.

The vector used in an integration method is preferably a plasmid. Aselectable marker may be included to allow for ease of identification ofdesired recombinant microorgansims. Additionally, as will be appreciatedby one of skill in the art, the vector is preferably one which can beselectively integrated into the chromosome. This can be achieved byintroducing an inducible origin of replication, for example, atemperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

Integration by the Campbell-type mechanism can take place in the 5′flanking region of the protease gene, resulting in a protease positivestrain carrying the entire plasmid vector in the chromosome in theserine protease locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

Another method of inactivating the naturally occurring serine proteasegene is to mutagenize the chromosomal gene copy by transforming agram-positive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal serine protease gene can be replaced witha mutant gene by homologous recombination.

The present invention encompasses host cells having additional proteasedeletions or mutations, such as deletions or mutations in apr, npr, epr,mpr and others known to those of skill in the art. U.S. Pat. No.5,264,366 discloses Bacillus host cells having a deletion of apr andnpr, U.S. Pat. No. 5,585,253 discloses Bacillus host cells having adeletion of epr; Margot et al., 1996, Microbiology 142: 3437-3444disclose host cells having a deletion in wpr and EP patent 0369817discloses Bacillus host cells having a deletion of mpr.

III. Production of Serine Protease

For production of serine protease in a host cell, an expression vectorcomprising at least one copy of nucleic acid encoding a gram-positivemicroorganism SP1, SP2, SP3, SP4 or SP5, and preferably comprisingmultiple copies, is transformed into the host cell under conditionssuitable for expression of the serine protease. In accordance with thepresent invention, polynucleotides which encode a gram-positivemicroorganism SP1, SP2, SP3, SP4 or SP5, or fragments thereof, or fusionproteins or polynucleotide homolog sequences that encode amino acidvariants of B. SP1, SP2, SP3, SP4 or SP5, may be used to generaterecombinant DNA molecules that direct their expression in host cells. Ina preferred embodiment, the gram-positive host cell belongs to the genusBacillus. In another preferred embodiment, the gram positive host cellis B. subtilis.

As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

Altered SP1, SP2, SP3, SP4 or SP5 polynucleotide sequences which may beused in accordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalent SP1,SP2, SP3, SP4 or SP5 homolog, respectively. As used herein a “deletion”is defined as a change in either nucleotide or amino acid sequence inwhich one or more nucleotides or amino acid residues, respectively, areabsent.

As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring SP1, SP2, SP3, SP4 or SP5.

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

The encoded protein may also show deletions, insertions or substitutionsof amino acid residues which produce a silent change and result in afunctionally SP1, SP2, SP3, SP4 or SP5 variant. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains theability to modulate secretion. For example, negatively charged aminoacids include aspartic acid and glutamic acid; positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,threonine, phenylalanine, and tyrosine.

The SP1, SP2, SP3, SP4 or SP5 polynucleotides of the present inventionmay be engineered in order to modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, eg, site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns or to change codon preference, for example.

In one embodiment of the present invention, a gram-positivemicroorganism SP1, SP2, SP3, SP4 or SP5 polynucleotide may be ligated toa heterologous sequence to encode a fusion protein. A fusion protein mayalso be engineered to contain a cleavage site located between the serineprotease nucleotide sequence and the heterologous protein sequence, sothat the serine protease may be cleaved and purified away from theheterologous moiety.

IV. Vector Sequences

Expression vectors used in expressing the serine proteases of thepresent invention in gram-positive microorganisms comprise at least onepromoter associated with a serine protease selected from the groupconsisting of SP1, SP2, SP3, SP4 and SP5, which promoter is functionalin the host cell. In one embodiment of the present invention, thepromoter is the wild-type promoter for the selected serine protease andin another embodiment of the present invention, the promoter isheterologous to the serine protease, but still functional in the hostcell. In one preferred embodiment of the present invention, nucleic acidencoding the serine protease is stably integrated into the microorganismgenome.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

V. Transformation

A variety of host cells can be used for the production of SP1, SP2, SP3,SP4 or SP5 including bacterial, fungal, mammalian and insects cells.General transformation procedures are taught in Current Protocols InMolecular Biology (vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, publishedMay 26, 1995).

In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more serine protease(s) of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the host cell. Suitable replicating plasmids forBacillus are described in Molecular Biological Methods for Bacillus, Ed.Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

In another embodiment, nucleic acid encoding a serine protease(s) of thepresent invention is stably integrated into the microorganism genome.Preferred host cells are gram-positive host cells. Another preferredhost is Bacillus. Another preferred host is Bacillus subtilis. Severalstrategies have been described in the literature for the direct cloningof DNA in Bacillus. Plasmid marker rescue transformation involves theuptake of a donor plasmid by competent cells carrying a partiallyhomologous resident plasmid (Contente et al., Plasmid 2:555-571 (1979);Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J.Bacteriol. 154(3):1077-1087 (1983); and Weinrauch et al., J. Bacteriol.169(3):1205-1211 (1987)). The incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

Transformation by protoplast transformation is described for B. subtilisin Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megaterium in Vorobjeva et al., (1980) FEMS Microbiol. Letters7:261-263; for B. amyloliquefaciens in Smith et al., (1986) Appl. andEnv. Microbiol. 51:634; for B. thuringiensis in Fisheret al., (1981)Arch. Microbiol. 139:213-217; for B. sphaericus in McDonald (1984) J.Gen. Microbiol. 130:203; and B. larvae in Bakhiet et al., (1985) 49:577.Mann et al., (1986, Current Microbiol. 13:131-135) report ontransformation of Bacillus protoplasts and Holubova, (1985) FoliaMicrobiol. 30:97) disclose methods for introducing DNA into protoplastsusing DNA containing liposomes.

VI. Identification of Transformants

Whether a host cell has been transformed with a mutated or a naturallyoccurring gene encoding a gram-positive SP1, SP2, SP3, SP4 or SP5,detection of the presence/absence of marker gene expression can suggestswhether the gene of interest is present However, its expression shouldbe confirmed. For example, if the nucleic acid encoding a serineprotease is inserted within a marker gene sequence, recombinant cellscontaining the insert can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem withnucleic acid encoding the serine protease under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the serine protease as well.

Alternatively, host cells which contain the coding sequence for a serineprotease and express the protein may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridization and proteinbioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

The presence of the cysteine polynucleotide sequence can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes, portionsor fragments of B. subtilis SP1, SP2, SP3, SP4 or SP5.

VII. Assay of Protease Activity

There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or calorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

VIII. Secretion of Recombinant Proteins

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram-positive host cell and detecting secretedproteins include, using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton R et al (1990, Serological Methods, a LaboratoryManual, APS Press, St Paul Minn.) and Maddox D E et al (1983, J Exp Med158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

IX. Purification of Proteins

Gram positive host cells transformed with polynucleotide sequencesencoding heterologous or homologous protein may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a serine protease of the presentinvention will be secreted into the culture media. Other recombinantconstructions may join the heterologous or homologous polynucleotidesequences to nucleotide sequence encoding a polypeptide domain whichwill facilitate purification of soluble proteins (Kroll D J et al (1993)DNA Cell Biol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein can be used to facilitate purification.

X. Uses of the Present Invention

Genetically Engineered Host Cells

The present invention provides genetically engineered host cellscomprising preferably non-revertable mutations or deletions in thenaturally occurring gene encoding one or more of SP1, SP2, SP3, SP4 orSP5 such that the proteolytic activity is diminished or deletedaltogether. The host cell may contain additional protease deletions,such as deletions of the mature subtilisn protease and/or mature neutralprotease disclosed in U.S. Pat. No. 5,264,366.

In a preferred embodiment, the host cell is genetically engineered toproduce a desired protein or polypeptide. In a preferred embodiment thehost cell is a Bacillus. In another preferred embodiment, the host cellis a Bacillus subtilis.

In an alternative embodiment, a host cell is genetically engineered toproduce a gram-positive SP1, SP2, SP3, SP4 or SP5. In a preferredembodiment, the host cell is grown under large scale fermentationconditions, the SP1, SP2, SP3, SP4 or SP5 is isolated and/or purifiedand used in cleaning compositions such as detergents. WO 95/10615discloses detergent formulation. A serine protease of the presentinvention can be useful in formulating various cleaning compositions. Anumber of known compounds are suitable surfactants useful incompositions comprising the serine protease of the invention. Theseinclude nonionic, anionic, cationic, anionic or zwitterionic detergents,as disclosed in U.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. Asuitable detergent formulation is that described in Example 7 of U.S.Pat. No. 5,204,015. The art is familiar with the different formulationswhich can be used as cleaning compositions. In addition, a serineprotease of the present invention can be used, for example, in bar orliquid soap applications, dishcare formulations, contact lens cleaningsolutions or products, peptide hydrolysis, waste treatment, textileapplications, as fusion-cleavage enzymes in protein production, etc. Aserine protease of the present invention may provide enhancedperformance in a detergent composition (as compared to another detergentprotease). As used herein, enhanced performance in a detergent isdefined as increasing cleaning of certain enzyme sensitive stains suchas grass or blood, as determined by usual evaluation after a standardwash cycle.

A serine protease of the present invention can be formulated into knownpowdered and liquid detergents having pH between 6.5 and 12.0 at levelsof about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. Thesedetergent cleaning compositions can also include other enzymes such asknown proteases, amylases, cellulases, lipases or endoglycosidases, aswell as builders and stabilizers.

The addition of a serine protease to conventional cleaning compositionsdoes not create any special use limitation. In other words, anytemperature and pH suitable for the detergent is also suitable for thepresent compositions as long as the pH is within the above range, andthe temperature is below the described serine protease denaturingtemperature. In addition, a serine protease of the present invention canbe used in a cleaning composition without detergents, again either aloneor in combination with builders and stabilizers.

One aspect of the invention is a composition for the treatment of atextile that includes a serine protease of the present invention. Thecomposition can be used to treat for example silk or wool as describedin publications such as RD 216,034; EP 134,267; U.S. Pat. No. 4,533,359;and EP 344,259.

Proteases can be included in animal feed such as part of animal feedadditives as described in, for example, U.S. Pat. Nos. 5,612,055;5,314,692; and 5,147,642.

Polynucleotides

A B. subtlis SP1, SP2, SP3, SP4 or SP5 polynucleotide, or any partthereof, provides the basis for detecting the presence of gram-positivemicroorganism polynucleotide homologs through hybridization techniquesand PCR technology.

Accordingly, one aspect of the present invention is to provide fornucleic acid hybridization and PCR probes which can be used to detectpolynucleotide sequences, including genomic and cDNA sequences, encodinggram-positive SP1, SP2, SP3, SP4 or SP5 or portions thereof.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto.

EXAMPLE I Preparation of a Genomic Library

The following example illustrates the preparation of a Bacillus genomiclibrary.

Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sau3Adigestion. Sau3A recognizes the 4 base pair site GATC and generatesfragments compatible with several convenient phage lambda and cosmidvectors. The DNA is subjected to partial digestion to increase thechance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II

The following example describes the detection of gram-positivemicroorganism SP1. The same procedures can be used to detect SP2, SP3,SP4 or SP5.

DNA derived from a gram-positive microorganism is prepared according tothe methods disclosed in Current Protocols in Molecular Biology, Chap. 2or 3. The nucleic acid is subjected to hybridization and/or PCRamplification with a probe or primer derived from SP1. A preferred probecomprises the nucleic acid section encoding conserved amino acidresidues.

The nucleic acid probe is labeled by combining 50 pmol of the nucleicacid and 250 mCi of [gamma ³²P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled probe is purified with Sephadex G-25 super fine resin column(Pharmacia). A portion containing 10⁷ counts per minute of each is usedin a typical membrane based hybridization analysis of nucleic acidsample of either genomic or cDNA origin.

The DNA sample which has been subjected to restriction endonucleasedigestion is fractionated on a 0.7 percent agarose gel and transferredto nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N. H.).Hybridization is carried out for 16 hours at 40 degrees C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1× saline sodium citrateand 0.5% sodium dodecyl sulfate. The blots are exposed to film forseveral hours, the film developed and hybridization patterns arecompared visually to detect polynucleotide homologs of B. subtilis SP1.The homologs are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Klenow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated intheir entirety.

1. A member of the genus Bacillus having a mutation or deletion of thegene encoding serine protease 3 (SP3), wherein said gene encoding serineprotease 3 comprises SEQ ID NO:6, said mutation or deletion resulting inthe inactivation of the SP3 proteolytic activity, and wherein saidmutation or deletion is present in the catalytic triad sequence of saidserine protease
 3. 2. The microorganism according to claim 1, whereinthe member is selected from the group consisting of B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus, and Bacillusthuringiensis.
 3. The microorganism of claim 1, wherein saidmicroorganism is capable of expressing a heterologous protein.
 4. Themicroorganism of claim 3, wherein said heterologous protein is selectedfrom the group consisting of hormones, enzymes, growth factors, andcytokines.
 5. The microorganism of claim 4, wherein said heterologousprotein is an enzyme.
 6. The microorganism of claim 5, wherein saidenzyme is selected from the group consisting of a proteases,carbohydrases, lipases, isomerases racemases, epimerases, tautomerases,or mutases, transferases, kinases and phophatases.
 7. An expressionvector comprising nucleic acid encoding SP3 having a mutation ordeletion, wherein said SP3 comprises SEQ ID NO:6, and said mutation ordeletion results in the inactivation of SP3 proteolytic activity, andwherein said mutation deletion is present in the catalytic triadsequence of said serine protease
 3. 8. A method for the production of aheterologous protein in a Bacillus host cell comprising the steps of (a)obtaining a Bacillus host cell comprising nucleic acid encoding saidheterologous protein wherein said host cell contains a mutation ordeletion in the gene encoding serine protease 3, wherein said serineprotease 3 comprises the nucleic acid sequence set forth in SEQ ID NO:6,and wherein said mutation or deletion is present in the catalytic triadsequence of said serine protease 3; and (b) growing said Bacillus hostcell under conditions suitable for the expression of said heterologousprotein.
 9. The method of claim 8, wherein said Bacillus cell isselected from the group consisting of Bacillus subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, and B. thuringiensis.
 10. The method of claim 9, wherein saidBacillus host cell further comprises a mutation or deletion in at leastone of the genes encoding apr, npr, epr, wpr and mpr.